Biophysical investigation of dynamics and topology of cell surface receptors
The primary immunological function of Major Histocompatibility Complex (MHC) molecules is to bind and "present" antigenic peptides on the surfaces of cells for recognition (binding) by the antigen-specific T cell receptors (TCRs) of lymphocytes. Differential structural properties of MHC class I and class II molecules account for their respective roles in activating different populations of T lymphocytes. Cytotoxic lymphocytes bind antigenic peptides presented by MHC class I molecules, whereas helper lymphocytes bind antigenic peptides presented by MHC class II molecules.
We study the two dimensional topology of several proteins transferrin receptor, IL-2 receptor, etc. in relation to MHC class I and II molecules, on different lymphoid cell lines and on human peripheral lymphocytes. We demonstrated the regulatory role of lateral organization of cell surface receptors on T cell activation. We develop and apply modern techniques for determination of different protein association patterns on cells in suspension or on cells bound to the extracellular matrix. Type I (Insulin-dependent) diabetes (T1D) is an autoimmune polygenic disease wherein the elevated amount of glucose persists in the bloodstream due to destruction of insulin-producing pancreatic β-cells. The etiology of the disease is complex, unresolved and advocates the contribution of both, environmental and genetic factors. The strongest genetic determinants reside in the Major Histocompatibility complex II (MHC II) core of chromosome 6 which constitutes a couple of genes (A and B encoding alfa and beta peptides) for each of HLA-DP, HLA-DQ and HLA-DR loci. Further on, these MHCII molecules, expressed by antigen presenting cells like B lymphocytes and dendritic cells, relay the antigen and activate CD4-positive T – cell mediated immune response. Regardless of the MHC II diasporas, HLA-DQ alleles have been the topmost diabetes susceptibility factor as also evident by the experiments in non obese diabetic (NOD) mice. Most importantly, HLA-DQ6 (DQA1*0102/ DQB1*0602 haplotype) molecule provides substantial protection against T1D / even nullifies the effect of high-risk HLA-DQ8 allele. Several propositions regarding the protection mechanism by this molecule have been described but still the answers remain abstruse. One such hypothesis states that the protective HLA-DQ6 allele could present antigen and interact diabetogenic T-cells in a different way compared to predisposing HLA-DQ2, -DQ8 alleles. To investigate the potential modifications, HLA-DQ6 molecules could introduce in the cell membrane, we selected the MHC II-deficient, Bare lymphocyte syndrome (BLS)-1 cell line for establishing the HLA-DQ6 expressing cell line typically named as tBLS-1 for transfected BLS-1. Since, MHCI, MHCII and Intercellular Adhesion Molecule 1 (ICAMI) are randomly distributed and often colocalized in the cell membrane; we expected that the new HLA-DQ6 molecule in tBLS-1 would be able to integrate and form clusters alongwith the MHCI and ICAM1 in the cell surface. These clusters at tBLS1 cell membrane would be different from the parent BLS1 cell membrane which could be then compared to JY lymphoblastoid cell line (normal expression of MHC and ICAM1) for analyzing the specific effects of HLA-DQ appearance.
We presented the proof of concept regarding the protein rearrangement on the cell membrane upon transfection of the HLA-DQ6 molecule. This particular organisation of proteins on cell surface might be crucial to HLA-DQ6 mediated antigen presentation and thus highlights the importance of surface protein dynamics in diabetes.
One of the other directions is the investigation of regulatory factors of homo and hetero associations (cluster formation) of MHC molecules on lymphoid cell lines. We study the effect of factors related to the composition of the plasma membrane (e.g. fatty acid content, ratio of cholesterol and phopholipids) and the potential regulatory roles of extracellular agents (e.g. exogen beta 2 microglobulin level).
We measure the approximate size of these protein clusters and the relationship between the expression level of the so called beta2 microglobulin free “free heavy chains” appearing on the surface of cells. The scope of our investigation is to assess the distribution of other MHC class I associated proteins (ICAM-1, IL-2, receptors) as well. According to our previous studies the function of MHC class I molecules is not restricted to the immune response; it might have a regulatory role in signal transduction of other receptors.
Composite SNOM image of an antibody labeled cell. The shear force topography and the fluorescence images were merged into a three dimensional representation in which height represents vertical displacement and superposed color the fluorescence intensity.

Composite SNOM image of an antibody labeled cell. The shear force topography and the fluorescence images were merged into a three dimensional representation in which height represents vertical displacement and superposed color the fluorescence intensity.